The polymerase chain reaction (PCR) is a primer extension reaction that provides a method to amplify a specific DNA or polynucleotide in vitro, generating thousands to millions of copies of a particular DNA sequence. PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints; and the detection and diagnosis of infectious diseases. Some of the variations of the basic PCR include quantitative real-time PCR (qPCR or RT-PCR), allele-specific PCR, asymmetric PCR, hot start PCR, reverse transcription PCR, multiplex-PCR, nested-PCR, ligation-mediated PCR, Intersequence-specific PCR, Thermal asymmetric interlaced PCR and touchdown-PCR. These PCR variations provide wide variety of uses for different purposes. For example, single-nucleotide polymorphisms (SNPs) (single-base differences in DNA) can be identified by allele-specific PCR, qPCR can provide a very high degree of precision in determining the number of copies amplified in the PCR reactions (Bartlett et al., “A Short History of the Polymerase Chain Reaction”, PCR Protocols, 2003).
Recently, High Resolution Melt (HRM) was added as a new molecular technique for high throughput mutation scanning (Zhou, L., et al., Clin. Chem. 51, 1770-1777, 2005). Mutation determination using FIRM is based on the dissociation of DNA, when exposed to an increasing temperature in the presence of fluorescent dyes interacting with double-stranded. DNA (see, for example U.S. Pat. No. 7,387,887; U.S. Pat. No. 7,582,429). There are numerous appropriate dyes disclosed in the art. The presence of a mutation leads to the formation of DNA heteroduplexes followed by a change in melting behavior. Thus, this “mutation scanning” technique detects the presence of sequence variations in target-gene derived PCR amplicons. In an HRM experiment, sample DNA is first amplified via real-time PCR in the presence of a High Resolution Melting Dye. Prominent examples of such dyes are disclosed in WO 2008/052742. After PCR, the successive melting experiment can be performed on the same Real Time Instrument, and analyzed with a respective Gene Scanning Software to identify sequence variants.
Classical swine fever (CSF), previously known as hog cholera is a highly contagious and multisystemic hemorrhagic disease affecting domestic and wild pigs that results in economic losses in the swine industry worldwide and is a notifiable disease to the Office International des Epizooties, according to the Terrestrial Animal Health Code (OIE, 2007). The causative agent is classical swine fever virus (CSFV) which is an enveloped, positive-sense, single stranded RNA virus, classified in the genus Pestivirus within the family Flaviviridae. At the genetic level, CSFV's can be divided into genotypes 1, 2 and 3, based on the partial sequences of the E2 and NS5B genes. Each genotype can be classified further into several sub-genotypes, referred to as 1.1, 1.2, and 1.3; 2.1, 2.2 and 2.3; and 3.1, 3.2, 3.3, and 3.4, respectively (Paton et al., Vet Microbiol. 73:137-157, 2000). In Asia, CSF epidemics are ubiquitous and genotypes 1, 2 and 3 have been isolated in several Asian countries.
CSFV can cause acute, sub-acute and chronic swine disease and poses a considerable threat to the swine industry worldwide causing severe economic losses. Control of CSF involves either eradication or vaccination, in which eradication is the preferred method of control in developed countries such as the Europian Union (EU) contributing to a significant amount of losses attributable to the slaughter of infected pigs. Vaccination on the other hand is the major control strategy in developing countries like China. The Hog Cholera Lapinized Virus (HCLV) (also known as the C strain) was developed in the mid-1950s and is widely used in China and many countries (Moorman et al., J Virol. 70:763-770, 1996). Massive vaccination with the HCLV vaccine makes it difficult to distinguish between wild-type and the HCLV-strain of CSFV in vaccinated swine herds (Van Oirshot, Vet Microbiol 96:367-384, 2003). Sub-clinical and asymptomatic infection with CSFV is universal (Moennig et al., Vet. J. 165, 11-20, 2003; Tu, Virus Res. 81, 29-37, 2001) making infections with wild-type CSFV not easily recognizable by farmers and veterinarians, and not detectable by general detection assays, such as virus isolation, antigen-capture ELISA, and fluorescent antibody tests.
Various diagnostic assays have been developed. However, they all have limitations. Virus isolation requires 6-12 days for confirmation of results; ELISA isn't very sensitive and often produces false negative results; real time PCR assays are faster and more sensitive but are limited by a high-risk of cross contamination and most importantly. None of these assays are able to distinguish between wild-type and the vaccines strains of CSFV. Previously, real-time RT-PCR assays for discriminating wild-type CSFV from the “Riems” vaccine-strain have been established in the EU (Leifer et al., J. Virol. Methods 158, 114-122, 2009; Leifer et al., J. Virol. Methods 166, 98-100, 2010; Leifer et al., J. Gen. Virol. 91, 2687-2697, 2010) and an assay for differentiating between wild-type and the “K-LOM’ vaccine-strain CSFV in Korea was developed (Cho et al., Can J Vet Res 70:226-229, 2006). In China, a two-step real time RT-PCR assay to distinguish wild-type CSFV from the HCLV-strain vaccine based on nucleotide differences at the probe binding site and a one-step real time RT-PCR assay (wt-rRT-PCR) using a minor groove binding (MGB) probe for detection of mutations in wild-types have been described.
It is unlikely that a universal detection assay could be used in all countries because different CSFV vaccine strains have been administered in different countries, e.g., “Riems” in the EU, “K-LOM in Korea and “HCLV” in China. Therefore, there is a need to develop an assay that is affordable, easy to execute and easy to interpret results to distinguish between wild-type and vaccine-type for Classical Swine Fever.
Infectious Bronchitis (IB) is a disease prevalent in all countries within poultry industry, with the incidence approaching 100% in most locations. It is the most economically important disease. In young chicks, respiratory disease or nephritis lead to mortalities, reduced weight gain and condemnation at processing, whereas in chickens of laying age, the disease is subclinical and results in reduced egg production and aberrant eggs (Ignjatovic et al., Archives of Virology, 2006). IB outbreaks continue to occur in vaccinated flocks mainly because it is thought to be caused by different serotypes, subtypes or variant of IBV, that are generated by nucleotide point mutations, insertions, deletions, or recombination of S1 genes.
The causative agent, Infectious Bronchitis Virus (IBV) belongs to the Coronaviridae family. It is an enveloped positive-sense, single stranded RNA virus, with a genome size of 27.6 kb in length. The first 20 kb encode the viral RNA-dependent RNA polymerase and proteases. The whole genome has at least ten open reading frames (ORF) from 5′ to 3′ and are as follows: 5′-1a-1b-S(S1, S2)-3a,b,c(E)-M-5a,b-N-Poly(A)-3′ encoding four structural proteins, including the spike glycoprotein (S), the membrane glycoprotein (M), the phosphorylated nucleocapsid protein (N) and the small membrane protein (E) (Mardani et al., Arch Virol. 155(10):1581-6, 2010).
IBV was first reported in the USA in 1930 and has since been reported in most countries throughout the four continents of America (Johnson and Marquardt, Avian Dis. 19:82-90, 1975), Europe (Capua et al., Zentralbl Veterinarmed B. 41:83-89, 1994; Cavanagh and Davis, Arch Virol 130:471-476, 1993; Gough et al., Vet Rec. 130:493-494, 1992), Asia (Wang et al., Avian Dis 41:279-282, 1997) and Australia (Ignjatovic and McWaters, J Gen Virol. 72:2915-2922, 1991; Lohr, Avian Dis. 20:478-482, 1976). In Malaysia, little is known about the prevalence of the disease. The first report of IB disease was as early as 1967 where the disease was mild and vaccination unwarranted (Chong et al., Second symposium on Scientific and Technological Research in Malaysia and Singapore, pp 73-83, 1967; Aziz et al., The 8th Veterinary Association Malaysia Scientific Congress, 23-25 Aug. 1996, Ipoh, pp 76-78). Variants have been present since at least 1979 (Lohr, Proceedings of the 1st International Symposium on Infectious Bronchitis, E F Kaleta & U. Heffels—Redmann (Eds), pp 70-75, 1988; de Wit et al. Avian Pathology. 40(3):223-235, 2011) with reports of a more virulent strain causing nephrosis-nephritis syndrome that lead to high mortality was first reported in 1980 (Heng et al., Kajian Veterinar. 12:1-8, 1980; Aziz 1996). Recent publications related to IBD in Malaysia dates back to year 2000, 2004 and 2009 with clinical reports of variant nepropathogenic IBV strains since 1995 (Maizan, Proceeding 12th FAVA and 14th VAM congress, 28-28 Aug. 2002, pp 116; Yap M. L et al. Proceeding VAM Congress 1-4 Sep. 2000; Arshad et al. J. Vet. Malaysia. 14 (1&2): 322002; Balkis et al. Proceedings VAM Congress 2004, Zulperi et al. Virus Genes. 38:383-391, 2009).
Worldwide, several different serotypes and genotypes of IBVs have been identified and new variants are still emerging. One of these new variant is QX-like IB. IB-QX has been circulating and reported in China since 2004 (Liu & Kong, Avian pathology, 33:321-327, 2004). The virus which is identified as QX has been predominantly associated with various forms of renal pathology. Other researchers have also reported similar strains in China (Liu et al., J of Gen Virol, 86:719-725, 2005). In 2007, Cuiping et al. (Vet. Microbio., 122:71, 2007) confirmed the data presented by Liu et al. (2005) reporting the isolation of nephropathogenic strains from vaccinated and unvaccinated chicken flocks between 2003 and 2005. Similar findings have also been reported in Russia and other parts of Europe (Bochkov et al., Avian Pathology, 35:379-393, 2006; Landman et al., Proceedings of the 14th World Veterinary Poultry Congress, 22-26 Aug. 2005, Istanbul, Turkey, pp 369).
It is unlikely that a universal detection assay could be used in all countries because different IBV vaccine strains have been administered in different countries. Therefore, there is a need to develop an assay that is affordable, easy to execute and easy to interpret results to distinguish between wild-type and vaccine-type for Infectious Bronchitis (IB).
Despite intensive vaccination programs, Newcastle disease virus (NDV) remains a constant threat to commercial poultry farms worldwide. NDV is a member of the order Mononegavirales, family Paramyxoviridae and genus Avulavirus. It is an enveloped virus which has a negative-sense, nonsegmented single-stranded RNA genome consisting of 15, 586 nucleotides. Its genome comprises of six genes: nucleoprotein (NP), phosphoprotein (P), matrix protein (M), fusion glycoprotein (F), hemagglutinin-neuraminidase (FIN), glycoprotein and large polymerase protein (L). Of the six genes found in NDV, its two membrane proteins, the F protein and the HN protein are most important in determination of its virulence.
The establishment of real-time PCR methods in recent years has brought significant development to molecular diagnostics of various infectious agents and has rapidly cut-down the turn-around-time for disease diagnostics for quick and accurate results for veterinary practitioners in the field and farmers. Many PCR assays have been described for the Real Time PCR assay for NDV detection and genotype differentiation using several different TaqMan probes or SYBR green (Wise et al., J. Clin Microbiol, 42:329-338, 2004). Tan et al. (J. Virol Method, 160:149-156, 2009) described a SYBR Green 1 real-time PCR for the detection and differentiation of NDV genotypes, however this assay required different primer pairs for detecting the different NDV genotypes and relied heavily on the analysis of the melting peaks to differentiate the three genotypes of NDV. Although SYBR Green 1 assay is the most cost effective and easiest form of real-time PCR to establish compared to other real-time detection formats, however, the major disadvantage is that the dye molecules binds with any double-stranded DNA that is present in the reaction mixture including non-specific PCR products or primer-dimers. Therefore, there is room for improvement in the current molecular diagnostic methods for rapid and conclusive results of NDV testing.